BR112019018410A2 - process for crystallizing 2'-fucosyl-lactose and related compositions - Google Patents

Abstract

the present invention relates to a process for crystallizing an oligosaccharide, particularly a human milk oligosaccharide, and, more particularly, 2'-fucosyl-lactose ("2'-fl"). this specification also refers to compositions (for example, crystalline products) produced using such a process.

Description

CROSS REFERENCE TO RELATED PATENT APPLICATIONS [001] This specification claims the priority of Provisional Patent Application N ^Q US 62 / 467,571 filed on March 6, 2017; European Patent Application 17167564.8 filed on April 21, 2017; and Provisional Patent Application N ^Q US 62 / 632,180 filed on February 19, 2018. The entire text of each of the patent applications referenced above is incorporated by reference into this specification.

FIELD [002] This specification relates to a process for crystallizing an oligosaccharide, particularly a human milk oligosaccharide, and, more particularly, 2'-fucosyl lactose (2'-FL). This specification also refers to compositions (for example, crystalline products) produced using such a process.

BACKGROUND [003] Human milk oligosaccharides are important for nutrition and therapeutic products. One of the most important human milk oligosaccharides is 2'-fucosyl-lactose (also known as 2'-O-fucosyl-lactose or 2'-FL). This oligosaccharide is the most abundant oligosaccharide found in human breast milk, and is believed to have several beneficial biological functions, including immune system-boosting, antiviral, antibacterial, prebiotic and brain development enhancing effects. Such benefits make 2'-FL a potentially attractive ingredient to be included in foods, dietary supplements and medications, and particularly infant formula.

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However, the development of economically feasible processes for large-scale production of 2'-FL remains a challenge.

[004] Many recent approaches to 2'-FL synthesis involve microbial fermentation processes, which produce 2'-FL from lactose. While there is success with this approach, such processes generally produce a complex product mix, which often includes other ingredients, such as lactose, oligosaccharides in addition to the desirable 2'-FL (eg, difucosillactose (DiFL)), monosaccharides, amino acids , polypeptides, proteins, monovalent and divalent salts, organic acids, nucleic acids, etc. Thus, there is still a need for reliable and economically feasible purification processes downstream that provide a usable 2'-FL product.

[005] A technique for purifying 2'-FL is crystallization. However, the commonly used cooling crystallization techniques generally fail to function with 2'-FL due to their unique crystallization properties. Although 2'-FL saturated water syrups exhibit low viscosity at low temperatures (which usually suggests that crystallization will be direct), even highly supersaturated aqueous 2'-FL syrups are generally stable at low temperatures.

[006] To overcome this problem, some of those skilled in the art have reported techniques that involve the addition of organic solvents to force crystallization. See, for example, Kuhn, R., et al., Chemische Berichte, volume 88 (8), pages 1,135 to 1,146 (1955); Kuhn, R., et al., Chemische Berichte, volume 89 (11), page 2,513 (1956); documents ^Q WO2011 / 150939; WO2015 / 188834; and WO201695924. There are, however, many potential disadvantages to using an organic solvent to crystallize 2'-FL. They include, for example, the

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3/57 cost of the organic solvent itself. Organic solvents also create harmful emissions, waste and / or pollution. Special equipment is often needed to recover the solvent after crystallization. In some cases, there are solvent losses associated with such solvent recovery. For some solvents, special equipment is also required due to the explosion or other safety reasons. And the use of solvents can also be a problem if the residual solvent remains in the crystalline product. Even residual levels of some organic solvents can have an adverse effect on taste, odor, color and / or food safety.

[007] Consequently, there is a continuing need for an effective, reliable and economically feasible downstream process to purify 2'-FL, particularly an aqueous crystallization that avoids the need for an organic solvent.

SUMMARY [008] Briefly, this specification discloses, in general, a process to crystallize 2'-FL, as well as compositions produced using such a process.

[009] In particular, this specification discloses, in part, a process for producing crystalline 2'-FL from an aqueous initial solution comprising 2'-FL and at least one other carbohydrate.

[010] In some embodiments, the process involves concentrating the initial solution to a supersaturated state in relation to 2'FL, and then precipitating a 2'-FL crystal from the supersaturated solution while subjecting the supersaturated solution to a temperature greater than than 60 ° C. In this process, the supersaturated solution comprises at most 1% (by weight) of organic solvent during the precipitation of the 2'-FL crystal.

[011] In some modalities, the process comprises concentrating the initial solution to a supersaturated state in relation to 2'Petition 870190087008, of 05/09/2019, p. 10/33

4/57 fucosyl-lactose, and then precipitate a 2'-fucosyl-lactose crystal from the supersaturated solution while submitting the supersaturated solution to a temperature of at least 40 ° C. In this process, the initial solution has a dry solids content with a concentration of 2'-fucosyl ilose from 70 to 95% (where the percentage corresponds to a normalized peak area concentration obtained using high performance liquid chromatography) . In addition, the supersaturated solution comprises a maximum of 1% (by weight) of organic solvent during the precipitation of the 2'-fucosyl-lactose crystal.

[012] In some embodiments, the process comprises concentrating the initial solution to a supersaturated state in relation to 2'fucosyl-lactose, and then precipitating a 2'-fucosyl-lactose crystal from the supersaturated solution while subjecting the supersaturated solution to a temperature of at least 40 ° C. In this process, the initial solution has a dry solids content with a concentration of 2'-fucosyl ilactose less than about 98% (where the percentage corresponds to a normalized peak area concentration obtained using high performance liquid chromatography. performance). In addition, the supersaturated solution comprises a maximum of 1% (by weight) of organic solvent during the precipitation of the 2'-fucosyl-lactose crystal. [013] In some embodiments, the process comprises concentrating the initial solution to a supersaturated state in relation to 2'fucosyl-lactose, and then precipitating a 2'-fucosyl-lactose crystal from the supersaturated solution while subjecting the supersaturated solution to a temperature of at least 40 ° C. In this process, the initial solution has a dry solids content with a concentration of 2'-fucosyl ILactose less than about 95% (where the percentage corresponds to a normalized peak area concentration obtained using high performance liquid chromatography) performance). Furthermore, the supersaturated solution comprises a maximum of 1% (by weight) of

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5/57 organic solvent during the precipitation of the 2'-fucosyl-lactose crystal. [014] In some embodiments, the process comprises concentrating the initial solution to a supersaturated state in relation to 2'fucosyl-lactose, and then precipitating a 2'-fucosyl-lactose crystal from the supersaturated solution while subjecting the supersaturated solution to a temperature of at least 40 ° C. In this process, the supersaturated solution comprises at most 1% (by weight) of organic solvent during the precipitation of the 2'-fucosyl-lactose crystal. In addition, the crystalline 2'-fucosyl lactose product has a melting point of about 230 to about 239 ° C (as determined with a heating rate of 1 ° C / min using the melting point method capillary of European Pharmacopoeia).

[015] In some embodiments, the process comprises concentrating the initial solution to a supersaturated state in relation to 2'fucosyl-lactose, and then precipitating a 2'-fucosyl-lactose crystal from the supersaturated solution while subjecting the supersaturated solution to a temperature of at least 40 ° C. In this process, the supersaturated solution comprises at most 1% (by weight) of organic solvent during the precipitation of the 2'-fucosyl-lactose crystal. In addition, the crystalline 2'-fucosyl-lactose product exhibits X-ray powder diffraction reflection, based on a measurement using CuKa radiation, at 16.98 ± 0.20, 13.65 ± 0 , 20 and 18.32 ± 0.20 angles 2Θ.

[016] This specification also discloses, in part, a process for producing amorphous 2'-FL. The process comprises producing crystalline 2'-FL according to the crystallization process described above (or elsewhere in this specification), dissolving the crystalline 2'-FL in a solvent (eg water) to form a solution of 2 Purified '-FL, and precipitating amorphous 2'-FL from the purified 2'FL solution.

[017] This specification also discloses, in part,

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6/57 processes to produce a food, dietary supplement or medicine.

[018] In some embodiments, the process for producing the food, dietary supplement or medication comprises producing crystalline 2'-FL according to the crystallization process described above (or elsewhere in this specification), and then mixing the Crystalline 2'-FL with one or more ingredients suitable for the food, dietary supplement or medication.

[019] In some embodiments, the process for producing the food, dietary supplement or medication comprises producing crystalline 2'-FL according to the crystallization process described above (or elsewhere in this specification), dissolving the 2'-FL crystalline in a solvent (eg water), and mix the dissolved 2'-FL with one or more ingredients suitable for the food, dietary supplement or medication.

[020] In some embodiments, the process for producing the food, dietary supplement or medication comprises producing amorphous 2'-FL, as described above (or elsewhere in this specification), and then mixing the amorphous 2'-FL with one or more ingredients suitable for the food, dietary supplement or medicine.

[021] In some embodiments, the process for producing the food, dietary supplement or medication comprises producing the amorphous 2'-FL as described above (or elsewhere in this specification), dissolving the amorphous 2'-FL in a solvent ( for example, water), and mix the dissolved 2'-FL with one or more ingredients suitable for the food, dietary supplement or medication.

[022] This specification also discloses, in part, processes for producing infant formula.

[023] In some embodiments, the process for producing formula

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Infant 7/57 comprises producing crystalline 2'-FL according to the crystallization process described above (or elsewhere in this specification), and then mixing crystalline 2'-FL with one or more infant formula ingredients.

[024] In some embodiments, the process for producing infant formula comprises producing crystalline 2'-FL according to the crystallization process described above (or elsewhere in this specification), dissolving the crystalline 2'-FL in a solvent ( eg water), and mix the dissolved 2'-FL with one or more infant formula ingredients.

[025] In some embodiments, the process for producing an infant formula comprises producing amorphous 2'-FL as described above (or elsewhere in this specification), and then mixing amorphous 2'-FL with one or more ingredients of infant formula.

[026] In some embodiments, the process for producing an infant formula comprises producing the amorphous 2'-FL as described above (or elsewhere in this specification), dissolving the amorphous 2'-FL in a solvent (for example, water ), and mix the dissolved 2'-FL with one or more infant formula ingredients.

[027] This specification also discloses, in part, a crystalline 2'-FL product obtained from a crystallization process described above (or elsewhere in this specification).

[028] This specification also discloses, in part, amorphous 2'-FL obtained from a process described above (or elsewhere in this specification) to produce amorphous 2'-FL.

[029] This specification also discloses, in part, a food, dietary supplement or medicine prepared by a process to produce a food, dietary supplement or medicine described above (or elsewhere in this report

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8/57 descriptive).

[030] This specification also discloses, in part, an infant formulation obtained from a process to produce an infant formula described above (or elsewhere in this specification).

[031] The additional benefits of the teachings in this specification will be evident to a person skilled in the art from reading this specification.

DETAILED DESCRIPTION [032] The detailed description is intended to recognize others skilled in the art with the Applicant's invention, its principles, and its practical application so that others skilled in the art can adapt and apply the invention in its various forms, since they can be better suited to the needs of a particular use. The detailed description and its specific examples, while indicating certain modalities, are intended for illustration purposes only. This specification, therefore, is not limited to the described modalities, and can be modified in several ways.

[033] This specification refers to a process for crystallizing 2'-FL from a solution containing 2'-FL. In general, this process comprises bringing a solution containing 2'-FL to a supersaturated state in relation to 2'-FL, and then crystallizing the 2'-FL from the solution by evaporation until a crystallization mass is obtained , which has a crystal yield of at least about 1% with respect to 2'-FL.

[034] In some embodiments, the solution from which 2'-FL is crystallized comprises a natural source of 2'-FL, such as, for example, animal milk (for example, human milk) or a composition derived from milk animal. In some embodiments, the solution from which 2'-FL is crystallized is produced by chemical synthesis. In

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9/57 In other modalities, the solution containing 2'-FL is obtained from a microbial fermentation process. For example, the source of 2'FL may be a fermentation broth obtained by microbial fermentation using a recombinant microorganism, such as bacteria or yeast. In some such modalities, the microorganism is a yeast. In other modalities, the microorganism is a bacterium. In some embodiments, the microorganism is Escherichia coli. In some embodiments, fermentation takes place in a chemically defined medium.

[035] Before crystallization, the source of 2'-FL (eg fermentation broth) can be subjected to one or more purification processes. In some embodiments, for example, the source of 2'-FL (for example, fermentation broth) is subjected to centrifugation, sedimentation or one or more other processes to remove cell biomass. In some embodiments, the source of 2'-FL (eg fermentation broth) is subjected to ultrafiltration. This can be useful, for example, to remove large cells and biomolecules, such as proteins, nucleic acids and lipopolysaccharides. In some embodiments, the source of 2'-FL (for example, fermentation broth) is subjected to nanofiltration. This can be useful, for example, to concentrate 2'-FL by reducing water while removing minerals and several small biomolecules. In some embodiments, the source of 2'FL (for example, fermentation broth) is subjected to chromatographic separation, such as gel filtration chromatography. In some embodiments, the source of 2'-FL (eg fermentation broth) is subjected to microfiltration. This can be useful, for example, to remove microbiological contamination. In some embodiments, the source of 2'-FL (for example, fermentation broth) is subjected to cation exchange, anion exchange, ion exchange resin, mixed bed ion exchange and / or electrodialysis. They can be useful, for example,

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10/57 to remove small charged molecules, salts and trace metals. In some embodiments, the source of 2'-FL (for example, fermentation broth) is subjected to discoloration, by contact, for example, with powdered activated carbon or carbon filtration. In some embodiments, the source of 2'-FL (for example, fermentation broth) is subjected to evaporation. This can be useful, for example, to concentrate 2'-FL by removing water. In some embodiments, the source of 2'-FL (for example, fermentation broth) is subjected to a combination of two or more of the above purification steps prior to crystallization. Such modalities may include using the steps in several orders, as well as repeating several steps at different points in the process.

[036] In some embodiments, for example, the source of 2'-FL is a fermentation broth, which is subjected to ultrafiltration, cation exchange, anion exchange, mixed bed ion exchange and powdered activated carbon before the solution is taken to a supersaturated state for crystallization. In some embodiments, the source of 2'-FL is a fermentation broth, which is subjected to centrifugation, ultrafiltration, cation exchange, anion exchange, mixed bed ion exchange and powdered activated carbon before the solution is brought to a state supersaturated for crystallization. In some embodiments, the source of 2'-FL is a fermentation broth, which is subjected to ultrafiltration, nanofiltration, cation exchange, anion exchange, mixed bed ion exchange and powdered activated carbon before the solution is brought to a state supersaturated for crystallization. In some embodiments, the source of 2'-FL is a fermentation broth, which is subjected to centrifugation, ultrafiltration, nanofiltration, cation exchange, anion exchange, mixed bed ion exchange and powdered activated carbon before the solution is brought to a supersaturated state for crystallization. In some embodiments, the source of 2'-FL is a fermentation broth, which is subjected to

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11/57 ultrafiltration, nanofiltration, optional microfiltration, optional ion removal (for example, ion exchange resin and / or electrodialysis), optional pre-concentration (for example, evaporation or nanofiltration), discoloration (for example, carbon filtration), microfiltration, and optional nanofiltration before the solution is brought to a supersaturated state for crystallization.

[037] In some embodiments, the concentration of 2'-FL (or purity) in the dry solids content of the syrup resulting from the pre-crystallization purification steps is less than 99%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is less than 98%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is less than about 95%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is less than about 91%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is less than about 90%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is less than about 85%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is less than about 80%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is less than about 75%.

[038] In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is at least about 50%. In some modalities, the

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The concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is at least about 55%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is at least about 60%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is at least about 65%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is at least about 70%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is at least about 75%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is at least about 80%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is at least about 85%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is at least about 90%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is at least about 95%. [039] In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is about 60 to about 95%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is about 60 to about 90%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is about 65 to about 95%. In

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In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is about 65 to about 90%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is about 70 to about 95%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is about 70 to about 90%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is about 70 to about 85%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is about 70 to about 80%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is about 70 to about 75%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is about 75 to about 95%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is about 80 to about 95%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is about 85 to about 95%. In some embodiments, the concentration of 2'-FL in the dry solids content of the syrup resulting from the pre-crystallization purification steps is about 90 to about 95%.

[040] The 2'-FL concentration percentages above correspond to the normalized peak area concentrations (or purities) obtained using high performance liquid chromatography (HPLC).

[041] In some modalities, 2'-FL is crystallized from

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14/57 a solution comprising 2'-FL and at least one other carbohydrate. For example, when the 2'-FL solution is derived from an enzymatic method or fermentative approach, the solution may contain, for example, a mixture of lactose complex, oligosaccharides in addition to 2'-FL (eg difucosyl-lactose (DiFL)), monosaccharides, amino acids, polypeptides, proteins, monovalent and divalent salts, organic acids, nucleic acids, etc. In some embodiments, the total concentration of carbohydrates other than 2'-FL in the solution is at least about 1%. In some embodiments, the total concentration of carbohydrates other than 2'-FL in the solution is at least about 2%. In some embodiments, for example, the total concentration of carbohydrates other than 2'-FL in the solution is at least about 5%. In some embodiments, the concentration of DiFL in the solution is at least about 1%. In some embodiments, the concentration of DiFL in the solution is at least about 2%. In some embodiments, the concentration of DiFL in the solution is at least about 5%. In some embodiments, the concentration of DiFL in the solution is at least about 7%. In some embodiments, the concentration of DiFL in the solution is about 1 to about 20%. In some embodiments, the concentration of DiFL in the solution is about 2 to about 20%. In some embodiments, the concentration of DiFL in the solution is about 2 to about 18%. In some embodiments, the concentration of DiFL in the solution is about 5 to about 18%. In some embodiments, the concentration of DiFL in the solution is about 2 to about 10%. In some embodiments, the concentration of DiFL in the solution is about 5 to about 10%. In some embodiments, the concentration of DiFL in the solution is about 10 to about 18%. In some embodiments, the concentration of lactose in the solution is at most about 0.1%. In some embodiments, the concentration of lactose in the solution is at least about 0.1%. In some modalities, the concentration of

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15/57 lactose in the solution is at least about 2%. In some embodiments, the concentration of lactose in the solution is at least about 5%. In some embodiments, the concentration of lactose in the solution is about 0.1 to about 15%. In some embodiments, the concentration of lactose in the solution is about 0.1 to about 0.5%. In some embodiments, the concentration of lactose in the solution is about 1 to about 12%. In some embodiments, the concentration of lactose in the solution is about 2 to about 12%. In some embodiments, the concentration of lactose in the solution is about 5 to about 12%. In some embodiments, the concentration of lactose in the solution is about 2 to about 15%. In some embodiments, the concentration of lactose in the solution is about 5 to about 15%. These percentages correspond to normalized peak area concentrations (or purities) obtained using HPLC.

[042] In general, the solution is brought to a supersaturated state before crystallization. In some embodiments, the solution leads to a 2'-FL supersaturation greater than about 1.0. In some embodiments, the solution is brought to a 2'-FL supersaturation of about 1.05 to about 1.8. In some embodiments, the solution is brought to 2'-FL supersaturation from about 1.1 to about 1.7. In some embodiments, the solution is brought to a 2'-FL supersaturation of about 1.1 to about 1.5. In some embodiments, the solution is brought to a 2'-FL supersaturation of about 1.2 to about 1.7. In some embodiments, the solution is brought to a 2'-FL supersaturation of about 1.3 to about 1.7. In some embodiments, the solution is brought to a 2'-FL supersaturation of about 1.3 to about 1.5. In this specification, unless stated otherwise, the terms supersaturation and supersaturation correspond to a dimensionless ratio of the measured 2'-FL content of the 2'-FL solubility, in which the ratio is calculated from the equation a

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16/57 follow:

2'-FL content in solution s = solubility of 2'-FL at the same temperature [043] Here, s is supersaturation, and the unit of measurement for the content of 2'-FL and 2'-FL the solubility is g / 100 g of water. The solubility of 2'FL at different temperatures is provided in Example 15.

[044] In some embodiments, the solution is brought to a supersaturated state by increasing the dry solids content to at least about 50% (by weight). In some such embodiments, the dry solids content is increased to at least about 55% (by weight). In some such embodiments, the dry solids content is increased to at least about 60% (by weight). In some such embodiments, the dry solids content is increased to at least about 65% (by weight). In some such embodiments, the dry solids content is increased to a maximum of about 80% (by weight). In some such embodiments, the dry solids content is increased to a maximum of about 78% (by weight). In some such embodiments, the dry solids content is increased to a maximum of about 75% (by weight). In some such embodiments, the dry solids content is increased from about 50 to about 80% (by weight). In some embodiments, the dry solids content is increased from about 55 to about 78% (by weight). In some embodiments, the dry solids content is increased from about 60 to about 75% (by weight). In some embodiments, the dry solids content is increased from about 65 to about 75% (by weight). In some such embodiments, the dry solids content is increased to about 60% (by weight).

[045] In some embodiments, the solution is brought to a supersaturated state in relation to 2'-FL by evaporation. In some such

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17/57 modalities, evaporation (or at least a portion of it) is performed at atmospheric pressure. In some embodiments, evaporation (or at least a portion of it) is performed at a lower pressure. In some embodiments, evaporation (or at least a portion of it) is carried out at a pressure of at most about 2.5 x 10 ⁴ Pa (250 mbar). In some embodiments, evaporation (or at least a portion thereof) is carried out at a pressure of about 1.2 x 10 ⁴ (120) to about 2.5 x 10 ⁴ Pa (250 mbar). In some embodiments, evaporation (or at least a portion thereof) is carried out at a pressure of about 1.3 x 10 ⁴ Pa (130) to about 2.2 x 10 ⁴ Pa (220 mbar). In some embodiments, evaporation (or at least a portion of it) is carried out at a pressure of about 1.5 x 10 ⁴ Pa (150) to about 2.1 x 10 ⁴ Pa (210 mbar) [046] In general, the temperature at which evaporation is carried out will depend, for example, on the pressure. In some embodiments, the evaporation temperature is the boiling temperature of the solution under pressure. In some embodiments, the evaporation temperature is at least about 40 ° C. In some embodiments, the evaporation temperature is at least about 45 ° C. In some embodiments, the evaporation temperature is at least about 50 ° C. In some embodiments, the evaporation temperature is at least about 55 ° C. In some embodiments, the evaporation temperature is at least about 60 ° C. In some embodiments, the evaporation temperature is greater than 60 ° C. In some embodiments, the evaporation temperature is at most about 100 ° C. In some embodiments, the evaporation temperature is at most about 90 ° C. In some embodiments, the evaporation temperature is at most about 80 ° C. In some embodiments, the evaporation temperature is at most about 75 ° C. In some modalities, the

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18/57 evaporation temperature is at most about 70 ° C (while temperatures greater than about 70 ° C can be advantageous for evaporation, the use of such temperatures can cause undesirable product degradation and / or color formation) . In some embodiments, the evaporation temperature is about 45 to about 80 ° C. In some embodiments, the evaporation temperature is about 50 to about 80 ° C. In some embodiments, the evaporation temperature is about 55 to about 80 ° C. In some embodiments, the evaporation temperature is about 60 to about 80 ° C. In some embodiments, the evaporation temperature is greater than 60 to a maximum of about 80 ° C. In some embodiments, the evaporation temperature is about 60 to about 75 ° C. In some embodiments, the evaporation temperature is greater than 60 to a maximum of about 75 ° C. In some embodiments, the evaporation temperature is about 65 to about 75 ° C. In some embodiments, the evaporation temperature is about 40 to about 70 ° C. In some embodiments, the evaporation temperature is about 45 to about 70 ° C. In some embodiments, the evaporation temperature is about 50 to about 70 ° C. In some embodiments, the evaporation temperature is about 55 to about 70 ° C. In some embodiments, the evaporation temperature is about 60 to about 70 ° C. In some embodiments, the evaporation temperature is greater than 60 to a maximum of about 70 ° C.

[047] In some embodiments, seeding is used to initiate the formation of 2'-FL crystals from the supersaturated solution. In some of these modalities, sowing is done by adding seed crystals to the supersaturated solution. In some such embodiments, the seed crystals comprise a particulate 2'-FL powder. In some embodiments, the seed crystals comprise anhydrous 2'-FL seed crystals.

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19/57 [048] Seed crystals can be made by several processes, including, for example, those discussed in this specification. In some embodiments, the dried seeds are ground to obtain a smaller particle size. The seed crystals can also be used in suspension form.

[049] The desirable amount of seed crystals may depend, for example, on the size of the seed crystals.

[050] In some embodiments, the amount of dry seed crystals used is about 0.001 to about 1% (by weight) of particulate 2'-FL, based on the 2'-FL of the crystallization mass.

[051] In some embodiments, a seed crystal suspension is used, and the amount of seed crystals in the suspension is about 1 to about 30% (by weight) of particulate 2'-FL, based on 2 '-FL of the crystallization mass.

[052] In some embodiments, crystallization is initiated without adding 2'-FL seed crystals to the supersaturated solution. In some such modalities, for example, sowing is carried out using spontaneous sowing or ultrasonic sowing.

[053] In general, initiation of crystallization (for example, the addition of seed crystals) is performed when adequate supersaturation is achieved. In some embodiments, initiation of crystallization (for example, the addition of seed crystals) is performed when the supersaturation of 2'-FL is greater than about 1.0. In some embodiments, initiation of crystallization (for example, addition of seed crystals) is performed when supersaturation of 2'-FL is about 1.05 to about 1.8. In some embodiments, initiation of crystallization (for example, addition of seed crystals) is performed when the 2'-FL supersaturation is from about 1.1 to about 1.7. In some embodiments, initiation of crystallization (for example, addition of seed crystals) is performed when the

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20/57 supersaturation of 2'-FL is about 1.1 to about 1.5. In some embodiments, initiation of crystallization (for example, addition of seed crystals) is performed when supersaturation of 2'-FL is about 1.2 to about 1.7. In some embodiments, initiation of crystallization (for example, addition of seed crystals) is performed when supersaturation of 2'-FL is 1.3 to about 1.7. In some embodiments, initiation of crystallization (for example, addition of seed crystals) is performed when supersaturation of 2'-FL is about 1.3 to about 1.5.

[054] In some embodiments, initiation of crystallization (for example, the addition of seed crystals) is carried out when the dry solids content of the syrup is at least about 60% (by weight). In some embodiments, initiation of crystallization (for example, the addition of seed crystals) is carried out when the dry solids content of the syrup is at least about 65% (by weight). In some embodiments, initiation of crystallization (for example, the addition of seed crystals) is carried out when the dry solids content of the syrup is at most about 80% (by weight). In some embodiments, initiation of crystallization (for example, the addition of seed crystals) is carried out when the dry solids content of the syrup is at most about 75% (by weight). In some embodiments, initiation of crystallization (for example, addition of seed crystals) is carried out when the dry solids content of the syrup is about 60 to about 80% (by weight). In some embodiments, initiation of crystallization (for example, addition of seed crystals) is carried out when the dry solids content of the syrup is about 65 to about 80% (by weight). In some embodiments, initiation of crystallization (for example, addition of seed crystals) is carried out when the dry solids content of the syrup is about 65 to about 77% (by weight). In some

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In 21/57 embodiments, the initiation of crystallization (for example, addition of seed crystals) is carried out when the dry solids content of the syrup is about 65 to about 75% (by weight). In some embodiments, initiation of crystallization (for example, addition of seed crystals) is carried out when the dry solids content of the syrup is about 65 to about 70% (by weight). In some embodiments, initiation of crystallization (for example, addition of seed crystals) is carried out when the dry solids content of the syrup is about 70 to about 76% (by weight).

[055] Once crystallization is initiated, crystallization can generally be carried out at the boiling point of the solution.

[056] In some embodiments, at least a portion (or all) of the crystallization is carried out at atmospheric pressure.

[057] In some embodiments, at least a portion of the crystallization is performed at a pressure that is less than atmospheric pressure. In some such embodiments, crystallization (or at least a portion thereof) is carried out at a pressure of at most about 2.5 x 10 ⁴ Pa (250 mbar). In some embodiments, crystallization (or at least a portion thereof) is carried out at a pressure of about 50 to about 2.5 x 10 ⁴ Pa (250 mbar). In some embodiments, crystallization (or at least a portion thereof) is carried out at a pressure of about 5.0 x 10 ³ (50) to about 1.2 x 10 ⁴ Pa (120 mbar). In some embodiments, crystallization (or at least a portion thereof) is carried out at a pressure of about 1.2 x 10 ⁴ (120) to about 2.5 x 10 ⁴ Pa (250 mbar). In some embodiments, crystallization (or at least a portion thereof) is carried out at a pressure of about 1.3 x 10 ⁴ (130) to about 2.2 x 10 ⁴ Pa (220 mbar). In some embodiments, crystallization (or at least a portion thereof) is carried out at a pressure of about 1.5 x 10 ⁴ (150) to about 2.1 x 10 ⁴ Pa (210 mbar). In some

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22/57 embodiments, crystallization (or at least a portion thereof) is carried out at a pressure of about 1.7 x 10 ⁴ (170) to about 2.05 x 10 ⁴ Pa (205 mbar).

[058] In some embodiments, at least about 50% of the crystallization is performed at a subatmospheric pressure range discussed above. In some embodiments, at least about 75% of the crystallization is performed at the subatmospheric pressure range discussed above. In some embodiments, at least about 90% of the crystallization is performed at the subatmospheric pressure range discussed above. In some embodiments, at least about 95% of the crystallization is performed at the subatmospheric pressure range discussed above. In some embodiments, at least about 98% of the crystallization is performed at the subatmospheric pressure range discussed above. In some embodiments, at least about 99% of the crystallization is performed at the subatmospheric pressure range discussed above. In some embodiments, the entire crystallization is performed at a subatmospheric pressure range discussed above.

[059] In some embodiments, at least a portion of the crystallization is performed at the boiling temperature of the solution at pressure. In some embodiments, the temperature during crystallization does not drop below 20 ° C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of at least about 40 ° C. In some embodiments, at least a portion of the crystallization is performed at a temperature of at least about 45 ° C. In some embodiments, at least a portion of the crystallization is performed at a temperature of at least about 50 ° C. In some embodiments, at least a portion of the crystallization is performed at a temperature of at least about 55 ° C. In some embodiments, at least a portion of the crystallization is performed at

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23/57 a temperature of at least about 60 ° C. In some embodiments, at least a portion of the crystallization is carried out at a temperature greater than 60 ° C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of at most about 100 ° C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of at most about 90 ° C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of at most about 80 ° C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of at most about 75 ° C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of at most about 70 ° C (while temperatures greater than about 70 ° C can be advantageous for crystallization, the use of such temperatures can cause degradation product and / or color formation). In some embodiments, at least a portion of the crystallization is performed at a temperature of about 45 to about 80 ° C. In some embodiments, at least a portion of the crystallization is performed at a temperature of about 50 to about 80 ° C. In some embodiments, at least a portion of the crystallization is performed at a temperature of about 55 to about 80 ° C. In some embodiments, at least a portion of the crystallization is performed at a temperature of about 60 to about 80 ° C. In some embodiments, at least a portion of the crystallization is carried out at a temperature greater than 60 and at most about 80 ° C. In some embodiments, at least a portion of the crystallization is performed at a temperature of about 60 to about 75 ° C. In some embodiments, at least a portion of the crystallization is performed at a temperature greater than 60 ° C and at most about 75 ° C. In some modalities, at least one

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24/57 portion of the crystallization is performed at a temperature of about 65 to about 75 ° C. In some embodiments, at least a portion of the crystallization is performed at a temperature of about 40 to about 70 ° C. In some embodiments, at least a portion of the crystallization is performed at a temperature of about 45 to about 70 ° C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of about 50 to about 70 ° C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of about 55 to about 70 ° C. In some embodiments, at least a portion of the crystallization is performed at a temperature of about 60 to about 70 ° C. In some embodiments, at least a portion of the crystallization is carried out at a temperature greater than 60 and at most about 70 ° C.

[060] In some embodiments, at least about 50% of the crystallization is performed at a temperature range discussed above. In some embodiments, at least about 75% of the crystallization is performed at the temperature range discussed above. In some embodiments, at least about 90% of the crystallization is carried out at the temperature range discussed above. In some embodiments, at least about 95% of the crystallization is performed at the temperature range discussed above. In some embodiments, at least about 98% of the crystallization is carried out at the temperature range discussed above. In some embodiments, at least about 99% of the crystallization is performed at the temperature range discussed above. In some embodiments, the entire crystallization is carried out at a temperature range discussed above.

[061] In some embodiments, the pH of the crystallization is about 4 to about 11. In some embodiments, the pH for at least a portion of the crystallization is about 4 to about 8. In some

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25/57 embodiments, the pH during at least a portion of the crystallization is from about 5 to about 7. In some embodiments, the pH during at least a portion of the crystallization is from about 5 to about 6. In some embodiments , the pH during the entire crystallization is at most about 7. In some embodiments, the pH during the entire crystallization is about 5 to about 7. In some embodiments, the pH during the entire crystallization is about 5 to about 6.

[062] During crystallization, the crystal mass is typically mixed mechanically. In some embodiments, the mixture is optimally arranged to maximize homogeneity over the entire volume of the crystallization mass. This usually provides heat and mass transfer and small supersaturation gradients throughout the crystallization volume. In some embodiments, this is achieved with the use of traditional rotating mixer blades. In general, the intensity of the mixture is moderate to prevent excess formation of fine particles.

[063] During crystallization, the crystal suspension is generally subjected to boiling and evaporation until a sufficient degree of crystallization (yield, reduction in purity of 2'-FL of the mother liquor and / or crystal size) has been achieved. In some embodiments, evaporation crystallization is continued for about 1 to about 50 h. In some embodiments, evaporation crystallization is continued for about 2 to about 40 h. In some embodiments, evaporation crystallization is continued for about 2 to about 30 h. In some embodiments, evaporation crystallization is continued for about 2 to about 20 h. In some embodiments, evaporation crystallization is continued for about 2 to about 15 h. In some embodiments, evaporation crystallization is continued for about 2 to about 5 h.

[064] It is typically desirable to achieve a crystal yield

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26/57 2'-FL of about 1 to about 80% (where that yield is ο crystal yield of 2'-FL immediately after boiling). In some embodiments, the target yield is the maximum crystallization yield that allows for effective crystal separation, washing and drying. More specifically, low crystal yields are typically undesirable due to the fact that they create losses in manufacturing capacity and efficiency. However, increases in crystal yield cause the viscosity of the product suspension to increase. High viscosity, in turn, can create difficulty (and, in some cases, even prevent) the crystal separation. Thus, in some modalities, the target yield is the maximum yield corresponding to a viscosity that allows for cost-effective separation.

[065] In some embodiments, a crystallization mass is obtained, which has a crystal yield of about 1 to about 80% with respect to 2'-FL. In some embodiments, a crystallization mass is obtained, which has a crystal yield of about 30 to about 80% with respect to 2'-FL. In some embodiments, the crystallization mass is obtained which has a crystal yield of about 50 to about 80% with respect to 2'-FL. In some embodiments, the crystallization mass is obtained which has a crystal yield of about 55 to about 75% with respect to 2'FL. In some embodiments, the crystallization mass is obtained, which has a crystal yield of about 60 to about 75% with respect to 2'-FL. The crystal yields above refer to yields immediately after boiling.

[066] In some embodiments, the crystal size (i.e., the largest size of a crystal) of the main crystal population is at least about 1 pm. In some embodiments, the crystal size of the main crystal population is at least about 5 pm. In

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27/57 some modalities, the crystal size of the main crystal population is at least about 10 pm. In some embodiments, the crystal size of the main crystal population is at least about 20 pm. In some embodiments, the crystal size of the main crystal population is at least about 30 pm. In some embodiments, the crystal size of the main crystal population at most around 10,000 pm. In some embodiments, the crystal size of the main crystal population at most about 5,000 pm. In some embodiments, the crystal size of the main crystal population at most about 1,000 pm. In some embodiments, the crystal size of the main crystal population at most about 500 pm. In some embodiments, the crystal size of the main crystal population at most about 300 pm. In some embodiments, the crystal size of the main crystal population at most about 200 pm. In some embodiments, the crystal size of the main crystal population is in the range of about 1 to about 10,000 pm. In some embodiments, the crystal size of the main crystal population is in the range of about 10 to about 500 pm. In some embodiments, the crystal size of the main crystal population is in the range of about 30 to about 200 pm. In some embodiments, the crystal size of the main crystal population is in the range of about 30 to about 100 pm. In some embodiments, the crystal size of the main crystal population is in the range of about 100 to about 200 pm. As used in this specification, the term main crystal population is a visual judgment of a microscopic image of a crystallization mass. The outermost crystals that are abnormally small or abnormally large are ignored.

[067] In some embodiments, additional feed liquid is added to the 2'-FL solution simultaneously with crystallization

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28/57 by evaporation to increase the level of the 2'-FL solution in the crystallizer and / or to increase the dry substance content of the 2'-FL solution. In some embodiments, the additional feed liquid is added continuously. In some embodiments, the additional feed liquid is added in batches.

[068] In some embodiments, the solution containing 2'-FL is cooled simultaneously with crystallization. In some embodiments, crystallization comprises a combined boiling and cooling process. In such a process, the temperature can, for example, be lowered to a temperature of about 10 to about 20 ° lower than the sowing point temperature. In some such embodiments, the cooling rate is from about 10 / h to about 6 / h.

[069] In some embodiments, when evaporation crystallization is finished, the crystal mass is mixed before allowing the mass to cool. In some such embodiments, mixing is carried out under, for example, atmospheric pressure, at a temperature of about 40 to about 75 °. In some such embodiments, mixing is carried out under, for example, atmospheric pressure, at a temperature of about 40 to about 70 °. In some embodiments, mixing is carried out under, for example, atmospheric pressure, at a temperature of about 45 to about 70 °. In some embodiments, mixing is carried out under, for example, atmospheric pressure, at a temperature of about 50 to about 70 °. In some embodiments, mixing is carried out under, for example, atmospheric pressure, at a temperature of about 55 to about 70 °. In some embodiments, mixing is carried out under, for example, atmospheric pressure, at a temperature of about 60 to about 70 °. In some embodiments, mixing is carried out under, for example, atmospheric pressure, at a temperature greater than 60 and at most about 70 °. In some

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29/57 modalities, mixing is continued for about 0.5 to about 30 h. [070] In some embodiments, when evaporation crystallization is completed, the temperature of the crystallization mass is lowered to room temperature. In some embodiments, when the evaporation crystallization is completed, the temperature of the crystallization mass is lowered to about 20Ό. In some embodiments, a longer cooling period is used when the 2'-FL content is low.

[071] In some modalities, cooling is conducted for a period of about 3 days. In some modalities, cooling is conducted for a period of about 1 to about 3 days. In some modalities, cooling is conducted for a period of about 1 to about 30 h.

[072] In some embodiments, the cooling rate is from about 10 / h to about õO / h.

[073] In some embodiments, the apparent viscosity of the crystallization mass during evaporation crystallization is about 3 to about 200 Pa-s. In some embodiments, the apparent viscosity of the crystallization mass during evaporation crystallization is from about 5 to about 200 Pa-s. In some embodiments, the apparent viscosity of the crystallization mass during evaporation crystallization is from about 5 to about 100 Pa-s. In some embodiments, the apparent viscosity of the crystallization mass during evaporation crystallization is about 50 to about 100 Pa-s. In some embodiments, the apparent viscosity of the crystallization mass during evaporation crystallization is about 5 to about 50 Pa-s. In some embodiments, the apparent viscosity of the crystallization mass during evaporation crystallization is about 5 to about 30 Pa-s. A viscosity in these bands is generally adequate for effective separation of the crystallized material. If

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30/57 the crystallization mass becomes too viscous for effective separation, the viscosity of the crystallization mass can be decreased, for example, by decreasing the supersaturation of the crystallization mass. Supersaturation of the crystallization mass can be reduced, for example, by raising the temperature and / or diluting the crystallization mass with water or a solution containing 2'-FL.

[074] Crystals can be separated by, for example, centrifugation, filtration, decanting, etc. In some embodiments, the separation of the crystals comprises centrifugation.

[075] After separation, crystals are typically dried by, for example, contacting them with hot air. In some embodiments, the crystals are washed after separation. In some embodiments, the crystals are washed with water and then dried by, for example, contact with hot air. In some embodiments, the water content of the resulting crystal fraction is less than about 1% (by weight). In some embodiments, the water content of the resulting crystal fraction is less than about 0.5% (by weight). The water content can be measured by weight loss due to dryness or by Karl Fisher titration.

[076] In some modalities, the separation of solid and liquid (SLS) is performed in the crystalline product to remove micro-impurities, such as traces of protein and DNA, amino acids, carbohydrate impurities, trace elements, etc.

[077] In some embodiments, the 2'-FL content of the crystal fraction resulting from this process is at least about 85%. In some embodiments, the 2'-FL content of the resulting crystal fraction is at least about 90%. In some embodiments, the 2'-FL content of the resulting crystal fraction is at least about 91%. In some embodiments, the 2'-FL content of the resulting crystal fraction is at least about 92%. In some embodiments, the 2'-FL content of the crystal fraction

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The resulting 31/57 is at least about 93%. In some embodiments, the 2'-FL content of the resulting crystal fraction is at least about 94%. In some embodiments, the 2'-FL content of the resulting crystal fraction is at least about 95%. In some embodiments, the 2'-FL content of the resulting crystal fraction is at least about 96%. In some embodiments, the 2'-FL content of the resulting crystal fraction is at least about 97%. In some embodiments, the 2'-FL content of the resulting crystal fraction is at least about 98%. In some embodiments, the 2'-FL content of the resulting crystal fraction is at least about 99%. In some embodiments, the 2'-FL content of the resulting crystal fraction is at least about 99.5%. In some embodiments, the 2'-FL content of the resulting crystal fraction is at least about 99.9%. These percentages correspond to normalized peak area concentrations or purities obtained using HPLC.

[078] In some embodiments, the melting point of the crystals resulting from this process is about 230 to about 239 ° C. In some embodiments, the melting point of the crystals resulting from this process is from about 230 to about 232 ° C. In some embodiments, the melting point of the crystals resulting from this process is from about 230 to about 231 ° C. In some embodiments, the melting point of the crystals resulting from this process is from about 232 to about 239 ° C. In some embodiments, the melting point of the crystals resulting from this process is about 232 to about 238 ° C. In some embodiments, the melting point of the crystals resulting from this process is from about 233 to about 238 ° C. In some embodiments, the melting point of the crystals resulting from this process is from about 234 to about 237 ° C. In some embodiments, the melting point of the crystals resulting from this process is less than 236 ° C.

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32/57 modalities, the melting point of the crystals resulting from this process is at least about 233 ° C and less than 236 ° C. In some embodiments, the melting point of the crystals resulting from this process is at least about 234 ° C and less than 236 ° C. In some embodiments, the melting point of the crystals resulting from this process is about 233 ° C to 235 ° C. The above melting points correspond to a melting point determined with a heating rate of 1 ° C / min using the European Pharmacopoeia capillary melting point method.

[079] In some embodiments, the resulting crystals exhibit a powder X-ray diffraction spectrum discussed in the International Patent Application Publication. No. ^Q WO2011 / 150939 for the crystalline form cited in document No. ^Q WO2011 / 150939 as a polymorph of 2'-O-fucosyl-lactose crystalline II.

[080] In some embodiments, the resulting crystals exhibit X-ray powder diffraction reflection, based on a measurement using CuKa radiation, at 16.98 ± 0.20.13.65 ± 0.20 and 18.32 ± 0.20 angles 2Θ. In some embodiments, the resulting crystals exhibit X-ray powder diffraction reflection, based on a measurement using CuKa radiation, at 16.98 ± 0.20, 13.65 ± 0.20, 18, 32 ± 0.20 and 21.70 ± 0.20 2Θ angles. In some embodiments, the resulting crystals exhibit X-ray powder diffraction reflection, based on a measurement using CuKa radiation, at 16.98 ± 0.20, 13.65 ± 0.20, 18, 32 ± 0.20, 21.70 ± 0.20 and 15.22 ± 0.20 2Θ angles. In some embodiments, the resulting crystals exhibit X-ray powder diffraction reflection, based on a measurement using CuKa radiation, at 16.98 ± 0.20, 13.65 ± 0.20, 18, 32 ± 0.20, 21.70 ± 0.20, 15.22 ± 0.20 and 20.63 ± 0.20 2Θ angles. In some embodiments, the resulting crystals exhibit X-ray powder diffraction reflection, based on a measurement using CuKa radiation, the

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33/57

16.98 ± 0.20, 13.65 ± 0.20, 18.32 ± 0.20, 21.70 ± 0.20, 15.22 ± 0.20, 20.63 ± 0.20 and 11, 94 ± 0.20 angles 2Θ.

[081] In some embodiments, recrystallization is performed one or more times to increase the purity of 2'-FL. Recrystallization can be carried out, for example, by dissolving the 2'-FL crystals in water (typically deionized water), bringing the resulting solution to a supersaturated state in relation to 2'-FL (by means of, for example, evaporation) and crystallizing with the evaporation crystallization methodology described above.

[082] In some modalities, the yield is increased by carrying out the crystallization of the mother liquor produced by the initial crystallization. Such crystallization can be carried out, for example, by taking the mother liquor to a supersaturated state in relation to 2'-FL (by means of, for example, evaporation), and crystallizing using the evaporation crystallization methodology described above.

[083] In some embodiments, the crystalline product of 2'-FL from the process described in this specification is incorporated into a food (for example, human or pet food), dietary supplement or medication. In some embodiments, the crystalline product is incorporated into a human baby food (for example, infant formula). In some such modalities, 2'-FL is mixed with other ingredients of the food, dietary supplement or medication. When incorporated into an infant formula, for example, 2'-FL can be mixed with other infant formula ingredients such as skim milk, a carbohydrate source (eg lactose), a protein source (eg , whey protein concentrate), a fat source (eg superior oleic safflower oil), vitamins, minerals, etc. In some embodiments, the concentration of 2'-FL in the infant formula is approximately the same concentration as the concentration of 2'-FL generally present in milk

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34/57 human breast.

[084] In general, this process can be used to produce an anhydrous crystalline product. In some embodiments, the moisture content of the dried 2'-FL crystal product, as measured by Karl Fischer titration, is less than 1.0% (by weight). In some such embodiments, the moisture content of the dried 2'-FL crystal product is less than 0.9% (by weight). In some embodiments, the moisture content of the dried 2'-FL crystal product is less than 0.8% (by weight). In some embodiments, the moisture content of the dried 2'-FL crystal product is less than 0.7% (by weight). In some embodiments, the moisture content of the dried 2'-FL crystal product is less than 0.6% (by weight). In some embodiments, the moisture content of the dried 2'-FL crystal product is less than 0.5% (by weight). In some embodiments, the moisture content of the dried 2'-FL crystal product is less than 0.4% (by weight). In some embodiments, the moisture content of the dried 2'-FL crystal product is less than 0.3% (by weight). In some embodiments, the moisture content of the dried 2'-FL crystal product is less than 0.2% (by weight). In some embodiments, the moisture content of the dried 2'-FL crystal product is less than 0.1% (by weight). In some embodiments, no humidification is detected in the dried 2'-FL crystal product, as measured by Karl Fischer titration.

[085] In some embodiments, the 2'-FL crystalline product from the process described in this specification is converted into a different physical form for the intended application. For example, in some embodiments, the crystalline product is dissolved or suspended in water or another solvent to form a solution or suspension, which in turn is incorporated into (or otherwise used in the manufacture of) a food (for example , infant formula), dietary or

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35/57 medicine. In other embodiments, the crystalline product is dissolved in water or another solvent to form a solution, which is subsequently spray-dried to form an amorphous 2'-FL composition, which in turn is incorporated into (or, otherwise, used in the manufacture of) a food (for example, infant formula), dietetic or medicine.

[086] In some embodiments, when the 2'-FL product is incorporated into a food, dietary supplement or medication, the amount of 2'-FL is an amount that is effective to have a benefit, such as a prebiotic, antibacterial effect , antiviral, immune system enhancement and / or brain development enhancement.

[087] The crystallization described in this specification does not require an organic solvent to be present in the solution.

[088] In some embodiments, no alcohol (eg, methanol, ethanol, etc.) is added to the solution from which 2'-FL is crystallized. In some embodiments, no alcohol is added while the solution is taken to supersaturation in relation to 2'-FL. In some embodiments, no alcohol is added while evaporation is used to bring the solution to supersaturation in relation to 2'-FL. In some embodiments, no alcohol is added while 2'-FL crystallization occurs. In some embodiments, the total concentration of any alcohol present in the solution while the solution is brought to supersaturation in relation to 2'-FL is at most about 1% (by weight). In some embodiments, the total concentration of any alcohol present in the solution while evaporation is used to bring the solution to supersaturation in relation to 2'-FL is at most 1% (by weight). In some embodiments, the total concentration of any alcohol present in the solution while crystallization of 2'-FL occurs is at most about 1% (by weight). In some modalities,

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36/57 the total concentration of any alcohol present in the solution while the solution is brought to supersaturation in relation to 2'-FL is at most about 0.1% (by weight). In some embodiments, the total concentration of any alcohol present in the solution while evaporation is used to bring the solution to supersaturation in relation to 2'-FL is at most 0.1% (by weight). In some embodiments, the total concentration of any alcohol present in the solution while crystallization of 2'-FL occurs is at most about 0.1% (by weight). In some embodiments, the solution consists essentially of no alcohol while the solution is taken to supersaturation in relation to 2'-FL. In some embodiments, the solution consists essentially of no alcohol while evaporation is used to bring the solution to supersaturation in relation to 2'-FL. In some embodiments, the solution consists essentially of no alcohol while 2'-FL crystallization occurs. Here, the expression essentially consists of no alcohol means that the amount of alcohol present in the solution (if present) is insufficient to materially affect crystallization.

[089] In some embodiments, no organic acid solvent (eg, acetic acid) is added to the solution from which 2'-FL is crystallized. In some embodiments, no organic acid solvent is added while the solution is taken to supersaturation with respect to 2'-FL. In some embodiments, no organic acid solvent is added while evaporation is used to bring the solution to supersaturation with respect to 2'-FL. In some embodiments, no organic acid solvent is added while 2'-FL crystallization occurs. In some embodiments, the total concentration of any organic acid solvent present in the solution while the solution is brought to supersaturation in relation to 2'FL is at most about 1% (by weight). In some modalities, the

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37/57 total concentration of any organic acid solvent present in the solution while evaporation is used to bring the solution to supersaturation with respect to 2'-FL is at most 1% (by weight). In some embodiments, the total concentration of any organic acid solvent present in the solution while crystallization of 2'-FL occurs is at most about 1% (by weight). In some embodiments, the total concentration of any organic acid solvent present in the solution while the solution is brought to supersaturation with respect to 2'-FL is at most about 0.1% (by weight). In some embodiments, the total concentration of any organic acid solvent present in the solution while evaporation is used to bring the solution to supersaturation with respect to 2'-FL is at most 0.1% (by weight). In some embodiments, the total concentration of any organic acid solvent present in the solution while crystallization of 2'-FL occurs is at most about 0.1% (by weight). In some embodiments, the solution consists essentially of no organic acid solvent while the solution is brought to supersaturation with respect to 2'-FL. In some embodiments, the solution consists essentially of no organic acid solvent while evaporation is used to bring the solution to supersaturation in relation to 2'-FL. In some embodiments, the solution consists essentially of no organic acid solvent while 2'-FL crystallization occurs. Here, the expression essentially consists of no organic acid solvent means that the amount of organic acid solvent present in the solution (if present) is insufficient to materially affect crystallization.

[090] In some embodiments, no organic solvent is added to the solution from which 2'-FL is crystallized. In some embodiments, no organic solvent is added while the solution is taken to supersaturation in relation to 2'-FL. In some

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38/57 modalities, no organic solvent is added while evaporation is used to bring the solution to supersaturation in relation to 2'-FL. In some embodiments, no organic solvent is added while crystallization of 2'-FL occurs. In some embodiments, the total concentration of any organic solvent present in the solution while the solution is brought to supersaturation with respect to 2'-FL is at most about 1% (by weight). In some embodiments, the total concentration of any organic solvent present in the solution while evaporation is used to bring the solution to supersaturation with respect to 2'-FL is at most 1% (by weight). In some embodiments, the total concentration of any organic solvent present in the solution while 2'FL crystallization occurs is at most about 1% (by weight). In some embodiments, the total concentration of any organic solvent present in the solution while the solution is brought to supersaturation with respect to 2'-FL is at most about 0.1% (by weight). In some embodiments, the total concentration of any organic solvent present in the solution while evaporation is used to bring the solution to supersaturation with respect to 2'-FL is at most 0.1% (by weight). In some embodiments, the total concentration of any organic solvent present in the solution while 2'FL crystallization occurs is at most about 0.1% (by weight). In some embodiments, the solution consists essentially of no organic solvent while the solution is taken to supersaturation in relation to 2'-FL. In some embodiments, the solution consists essentially of no organic solvent while evaporation is used to bring the solution to supersaturation in relation to 2'-FL. In some embodiments, the solution consists essentially of no organic solvent while 2'-FL crystallization occurs. Here, the expression essentially consists of no organic solvent means that

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39/57 the amount of organic solvent present in the solution (if present) is insufficient to materially affect crystallization.

EXAMPLES [091] The following examples are illustrative only, and do not limit the rest of this specification in any way.

[092] In the examples below, the following definitions are used:

[093] A value identified by [1] refers to the normalized peak area purity or concentration obtained using HPLC (CarboPac PA100, Thermo Fisher Scientific, Waltham, MA USA). This is a percentage of peak area in relation to the total peak area.

[094] A value identified by [2] refers to the yield of 2'FL calculated from the purity values.

[095] A value identified by [3] refers to the dry solids (DS) content as measured by Karl Fischer titration.

[096] A value identified by [4] refers to the dry solids content calculated from a mass balance based on the initial dry solids content measured using Karl Fischer titration and the amount of water evaporated or added .

[097] A value identified by [5] refers to the moisture content measured in weight loss on drying.

[098] A value identified by [6] refers to a measurement of crystal size (that is, measurement of the largest dimension of a crystal) made from a microscopic image. The outermost crystals that are abnormally small or abnormally large are ignored.

[099] A value identified by [7] refers to a sugar color under the Commission's sugar color classification system

International for Uniform Methods of Sugar Analysis (ICUMSA).

[100] A value identified by [8] refers to a

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40/57 supersaturation in relation to 2'-FL defined as a ratio between the concentration of 2'-FL in solution (g / 100 g of water) and solubility of 2'-FL in water (g / 100 g of water) at the same temperature.

[101] A value identified by [9] is the melting point measured at a heating rate of 1 ° C / min using the European Pharmacopoeia capillary melting point method.

[102] A value identified by [10] refers to the normalized peak area purity or concentration obtained using HPLC (X-Bridge Amide HILIC column, Waters, Milford, MA USA). This is a percentage of peak area in relation to the total peak area.

[103] The abbreviation HPLC refers to high performance liquid chromatography.

[104] The abbreviation DiFL refers to difucosyl lactose.

Example 1: Illustration of 2'FL Seed Crystal Preparation [105] An aqueous feed solution, which had a concentration of 2'-FL of 90.4% [1], a concentration of DiFL of 8.9 % [1], and a lactose concentration of 0.2% [1], was evaporated at a DS of 84.4% [3] (Rotavapor R-151 evaporator). Ethanol (279 g) was then added to the syrup (415 g) by mixing the solution manually with an anchor shaker. The resulting solution was kept in a Rotavapor bottle rotating at 600 ° C for 2.3 h. During this time, water (10 g) was added twice (20 g in total) to dilute the solution. The first addition of water occurred 1.0 h after the addition of ethanol, and the second addition of water occurred 1.6 h after the addition of ethanol.

[106] A portion of the resulting syrup (306 g) was moved to a glass reactor. Ethanol (71 g) was added to 600. Crystals started to form. The crystallization solution was stirred at 600 for 1.2 h, cooled to 550 in 1 h, and additionally to 150 in 15 h with continuous stirring. After cooling, the crystal mass obtained

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41/57 continued to be stirred at 15Ό for 3.9 h.

[107] The crystal mass was centrifuged with a batch centrifuge that is 22.5 cm in basket diameter. For this centrifugation, 343 g of crystal mass was loaded into the centrifuge in the acceleration stage at a rotation speed of 500 rpm. The washing water (75 ml) was added at a rotation speed of 2,000 rpm and then the centrifugation was continued for 5 min at a rotation speed of 3500 rpm. The resulting centrifuged crystals (85 g) were dried in a heating chamber at 500 ° C for 3.4 h. The moisture content after drying was 0.8% [3].

[108] Crystals prepared according to the process illustrated here were used in Example 2. Before being used, the crystals were washed with ethanol, dried and then ground in a porcelain mill to break the crystal structure and provide irregularly shaped particles to start the secondary nuclear. The washed crystals had a 2'-FL purity of 94.0% [1].

Example 2: Preparation of Seed Crystals

Using Seed Crystals Prepared According to Example 1 [109] An aqueous feed solution, which had a concentration of 2'-FL of 90.7% [1], a concentration of DiFL of 9.3% [1 ], and a lactose concentration of 0.1% [1], was evaporated at a DS of 82.9% [3] (Rotavapor R-151 evaporator). The resulting syrup (683 g) was moved to a glass reactor, and seeded with 0.29 g of dry 2'-FL seed crystals (prepared according to Example 1) at a temperature of 55 °.

[110] The seeded syrup was stirred while kept at 55Ό. Ethanol was added in two portions: the first portion (610 g) was added 0.9 h after sowing in 0.3 h, and the second portion (221 g) was added 2.3 h after sowing. The crystallization solution was then cooled from 55Ό to 15Ό in 17 h during the gitation. after the

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42/57 cooling to 150 ° C, stirring was continued at 15 ° C for 0.9 h. The resulting crystal mass (1.416 g) was centrifuged with 100 ml of washing water (batch centrifuge, basket diameter 22.5 cm,

3,500 rpm, 5 min). A total of 557 g of crystal cake was obtained. Thereafter, 241 g were dried in a heating chamber at 60 ° C for 2.3 h. The moisture content after drying was 0.8% [3].

[111] Crystals prepared according to this process were, in turn, used as seed crystals in Example 3 below. Before being used, the crystals were washed with ethanol, dried and then ground in a porcelain mill. The washed crystals had a 2'-FL purity of 91.6% [1].

Example 3: Illustration of 2'-FL Crystallization [112] An aqueous feed solution, which had a 2'-FL concentration of 89.3% [1], a DiFL concentration of 9.5% [1 ], and a lactose concentration of <0.1% [1], was evaporated at a DS of 61.0% [4] (Rotavapor R-151 evaporator). The syrup (1.540 g) was then seeded with 0.5 g of dry 2'-FL seed crystals (preparation described in Example 2) at a temperature of 570 (1.01 supersaturation [8]). This resulted in very small crystal formation. Consequently, the seeded syrup was kept boiling at a temperature of 640 and a pressure of 1.3 x 10 ⁴ to 1.8 x 10 ⁴ Pa (130 to 180 mbar) for 2.8 h. The syrup was then seeded a second time with 0.5 g of dry 2'-FL seed crystals at a 67.2% DS [4] at a temperature of 650 (1.27 supersaturation [8] ). After the second sowing, crystallization occurred. Boiling crystallization was continued at a temperature of 650 ° C and a pressure of 1.75 x 10 ⁴ Pa (175 mbar) for 3.3 h.

[113] The crystal mass obtained, which had a DS of 72.8% [3], was allowed to cool to room temperature (21.50) and was then kept in a rotating one-day Rotavapor bottle to the other (18

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H). After 21.5 h of the first sowing, a small sample of the mass was centrifuged without washing (Hettich Rotanta 460R centrifuge,

2,500 rpm, 10 min) to determine the crystal content. The purity of 2'-FL of crystal cake was 96.7% [1] and the purity of 2'-FL of mother liquor was 85.6% [1]. This corresponds to 36% yield of 2'-FL [2]. The viscosity of the mass was 3.2 Pa-s.

[114] The crystal mass was heated to 65Ό, and boiling crystallization was continued under vacuum at a pressure of 1.7 x 10 ⁴ Pa (170 mbar) for 4.4 h until the DS of the crystal mass was 81, 5% [3]. A small sample of the mass was centrifuged without washing. This resulted in a 2'-FL crystal cake purity of 97.2% [1], a 2'-FL purity of mother liquor of 73.5% [1], a 2'-FL centrifuge yield of 72 % [2], and DS of mother liquor of 67.9% [3]. The crystal size of the main crystal population was 30 to 60 pm [6].

[115] The crystal mass was diluted to a DS of 79.5% [4] by adding deionized water, and then centrifuged with a batch centrifuge that is 22.5 cm in diameter. Here, 693 g of crystal mass was loaded into the centrifuge. The wash water (50 ml) was then added during the acceleration stage at a rotation speed of 2,000 rpm. The centrifugation time was continued for 5 min at a rotation speed of 3,500 rpm. The centrifugation yield of 2'-FL was 54% [2]. The purity of 2'-FL of mother liquor was 79.5% [1], and the moisture content of the non-dried cake 5.7% [5].

[116] The centrifugation crystal cake (274 g) was dried in a 60Ό heating chamber for 2 h and then cooled in a desiccator. The dried crystals had a 2'-FL purity of 100.0% [1], a melting point of 236.8 to 237.5 ° C [9], a color corresponding to 22 ICUMSA [7] and a humidification content of 0.3% [3].

[117] The centrifugation (30 g) non-dried crystal cake was

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44/57 washed with ethanol, dried and then ground in a porcelain mill to prepare seed crystals for Examples 5, 7, 8, 10, 12 and 13.

Example 4: Crystallization of 2'-FL from the Centrifuge Mother Liquor of Example 3 [118] The centrifuge mother liquor of Example 3, which had a 2'-FL concentration of 79.5% [1 ], a DiFL concentration of 17.9% [1], and lactose concentration of <0.1% [1], was evaporated at a temperature of 73Ό and a pressure of 2.0 x 10 ⁴ Pa (200 mbar) (Rotavapor R-151 evaporator). When the DS was 74.9% [4], the crystals formed by spontaneous nucleation (supersaturation 1.55 [8]). The syrup was diluted to a DS of 72.6% [4] and kept at 73Ό without vacuum for 1, 2 h. The syrup boiling was then continued at a temperature of 73Ό and a pressure of 1.75 x 10 ⁴ to 1.8 x 10 ⁴ Pa (175 to 180 mbar) for 4.8 h. The obtained crystal mass, which had a DS of 83.2% [3], was moved to a glass bottle and kept in a heating chamber at a temperature of 60Ό for 69 h. 74 h after the start of spontaneous nucleation, centrifuging a small sample of the unwashed mass (Hettich Rotanta 460R centrifuge, 2,500 rpm, 10 min) provided a crystalline cake that has a 2'-FL purity of 95.7% [1 ] and a mother liquor that has a 2'-FL purity of 51.5% [1]. This corresponds to a 2'-FL yield of 76% [2]. The crystal size of the main crystal population was 40 to 100 pm [6].

[119] The crystal mass (165 g) was centrifuged with a batchless washing centrifuge (basket diameter 22.5 cm,

3,500 rpm, 3 min). The yield of 2'-FL centrifugation was 76% [2]. The crystals centrifuged and dried (60Ό, 2.4 h) had a purity of 2'-FL of 93.2% [1] with a color of 157 ICUMSA [7] and humidification content of 0.5% [3 ]. The purity of 2'-FL of mother liquor was 53.9% [1] with a color of 609 ICUMSA [7].

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Example 5: Use of Simultaneous Fresh Feeding with Crystal Precipitation [120] An aqueous feed syrup, which had a 2'-FL concentration of 87.3% [1], a DiFL concentration of 10.1% [ 1], and a lactose concentration of 0.2% [1], was evaporated at a DS of 67.7% [4] (thin film evaporator Luwa NL3-210 / 1600/10). At that point, 1.149 g of the syrup was moved to a Rotavapor R-153 evaporator and seeded with 0.83 g of 2'-FL dry seed (crystals of Example 3) at a temperature of 61Ό (1.30 supersaturation [8 ]). The seeded syrup was boiled at a temperature of 65Ό and pressure of 1.5 x 10 ⁴ Pa at 1.65 x 10 ⁴ Pa (150 to 165 mbar) for 5.1 h while simultaneously adding fresh feed liquid (DS 58.3% [4], 1,443 g in total) to the crystal mass in small portions (approximately 30 to 120 g) at intervals of 5 to 40 min. After that, the DS of the crystal mass was 80.3% [3]. Centrifuging a small sample of the dough without washing (Hettich Rotanta 460R centrifuge, 2,500 rpm, 10 min) provided a crystalline cake that has a purity of 96.1% 2'-FL [1] and purity 2'- FL of mother liquor of 75.2% [1], which corresponds to a yield of 2'-FL of 64% [2]. The crystal size of the main crystal population was 30 to 60 pm [6].

[121] The crystal mass (1.332 g) was centrifuged with 75 ml of washing water (batch centrifuge, basket diameter 22.5 cm,

3,500 rpm, 5 min). The centrifugation yield of 2'-FL was 53% [2]. The 2'-FL purity of the centrifuged and dried crystals (60Ό, 8-1 7 h) was 97.8% [1]. The color was 67 ICUMSA [7] and the humidification content was 0.1% [3]. The purity of 2'-FL of mother liquor was 78.1% [1] and the color was 93 ICUMSA [7] ·

Example 6: Crystallization of 2'-FL from Centrifuge Mother Liquor of Example 5

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Use Addition of Water Simultaneously with Crystal Precipitation [122] The centrifuge mother liquor of Example 5, which had a 2'-FL concentration of 78.1% [1], a DiFL concentration of 17.6% [1], and a lactose concentration of 0.3% [1], was evaporated at a temperature of 67Ό and pressure of 2.0 x 10 ⁴ Pa (200 mbar) (Rotavapor R-151 evaporator). When the DS was 75.5% [4], the crystals formed by spontaneous nucleation (supersaturation 1.65 [8]). The syrup was diluted to a DS of 72.6% [4] and kept at 67Ό without vacuum for 1 h. Boiling the syrup was then continued at a temperature of 65 to 67Ό and the pressure of 2.0 x 10 ⁴ Pa (200 mbar) for 5.2 h while simultaneously adding small portions of deionized water (around 500 g in total) for the dough.

[123] After 6.2 h from the beginning of spontaneous nucleation, the DS of the crystal mass was 81.8% [3]. Centrifuging a small sample of the dough without washing (Hettich Rotanta 460R centrifuge, 2,500 rpm, 10 min) provided a crystalline cake that has a 2'-FL purity of 91.9% [1] and a mother liquor that has a 2'-FL purity of 59.0% [1]. This corresponds to a 2'-FL yield of 68% [2]. The crystal size of the main crystal population was 30 to 80 pm [6].

[124] The crystal mass (371 g) was centrifuged with 30 ml of washing water (batch centrifuge, basket diameter 22.5 cm, 3,500 rpm, 5 min). The centrifugation yield of 2'-FL was 54% [2]. The 2'-FL purity of the centrifuged and dried crystals (60Ό, 2 h) was 93.4% [1]. The color was 100 ICUMSA [7] and the humidification content was 0.9% [3]. The purity of 2'-FL of mother liquor was 65.4% [1] and the color was 287 ICUMSA [7] ·

Example 7: Illustration of 2'-FL Crystallization [125] An aqueous feed syrup, which had a 2'-FL concentration of 89.9% [1], a DiFL concentration of 8.5% [1 ], and a lactose concentration of <0.1% [1], was evaporated to a

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65.6% DS [3] (Luwa thin film evaporator NL3-210 / 1600/10). The resulting syrup (8141 g) was moved to a Rotavapor R153 evaporator and seeded with 2.7 g of 2'-FL dry seed (crystals of Example 3) at a temperature of 68Ό (1.17 supersaturation [8]) . The mixing was continued at this temperature for 1.2 h. After that, the seeded syrup was evaporated to a DS of 80.4% [3] in 5.3 h in three stages so that, in each stage, the syrup was first boiled under vacuum for 0.9 to 1 , 2 h (67 to 72Ό and 1.9 x 10 ⁴ to 2.0 x 10 ⁴ Pa (190 to 200 mbar)), followed by mixing at atmospheric pressure for 0.6 to 0.9 h (67 to 72 a) . The resulting crystal mass was moved to a 6 L mixing tank and kept at 69Ό overnight (7 pm). The crystallization progress was monitored by centrifuging (Hettich Rotanta 460R centrifuge, 2,500 rpm, 10 min) small samples of the mass without washing. The results are summarized in Table 1.

[126] The resulting crystal mass was centrifuged in 3 batches using an amount of washing water equal to 62 to 82 ml / kg of dough mass (batch centrifuge, basket diameter 22.5 cm,

3,500 rpm, 5 min). The moisture content of the combined non-dried cake was 4.4% [5]. The 2'-FL purity of the centrifuged and dried crystals (600, 2 h) was 100.0% [1]. The color was 462 I CUMSA [7] and the humidification content was 0.9% [3]. The crystal size of the main crystal population was 30 to 100 pm [6].

Table 1

Crystallization in terms of DS [3] or * [4], purity of 2'-FL [1] and centrifugation yield [2]

sowing time H crystal mass mother liquor crystal cake centrifugation performance 2'-FL % DS % 2’-FL % DS % 2’-FL % DS % 2’-FL % 2.7 71.1 · 89.9 72.0 90.6 4.8 76.1 · 89.9 70.7 86.0 95.8 98.7 34 6.5 80.4 89.9 69.8 80.2 95.6 98.1 59 25.0 80.4 89.9 66.0 75.8 95.1 97.2 71

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Example 8: Illustration of 2'-FL Crystallization

From Mother Liquor and Equipment Wash from Example 7 [127] The centrifuge mother liquor combined with diluted crystal mass recovered from washing the equipment from Example 7, which had a 2'-FL concentration of 87.4% [ 1], a DiFL concentration of 11.1% [1], and a lactose concentration of <0.1% [1], was evaporated to a DS of 65.1% [4] (Rotavapor R-153 evaporator ). The syrup (4.812 g) was seeded with 1.75 g of dry 2'-FL seed (crystals of Example 3) at a temperature of 65Ό (1.13 supersaturation [8]). The seeded syrup was then boiled at a temperature of 68 to 70Ό and pressure from 1.9 x 10 ⁴ to 1.95 x 10 ⁴ Pa (190 to 195 mbar) for 6.1 h. The DS of the resulting crystal mass was 80.8% [3]. Centrifuging a small sample of the dough without washing (Hettich Rotanta 460R centrifuge, 2,500 rpm, 10 min) provided a 2'FL of crystalline cake that has a purity of 96.5% [1] and a 2'-FL of liquor - mother that has a purity of 76.7% [1]. This corresponds to a 2'-FL yield of 60% [2].

[128] The crystal mass was kept in the rotating Rotavapor bottle at a temperature of 68Ό overnight (18 h). After 23 h of sowing, centrifuging a small sample of the unwashed mass (Hettich Rotanta 460R centrifuge, 2,500 rpm, 10 min) provided a crystalline cake that has a 2'-FL purity of 97.3% [1] and a 2'-FL purity of mother liquor of 69.7% [1]. This corresponds to a 2'-FL yield of 71% [2]. The crystal size of the main crystal population was 30 to 80 pm [6].

[129] The resulting crystal mass was centrifuged in two batches of washing water at an amount equal to 61 to 75 ml / kg of mass DS (batch centrifuge, basket diameter 22.5 cm, 3,500 rpm , 5 min). The moisture content of the combined non-dried cake was 3.7% [5]. The 2'-FL purity of the crystals

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49/57 centrifuged and dried (60Ό, 2.2 h) was 97.1% [1]. The color was 389 ICUMSA [7] and the humidification content was 0.5% [3].

Example 9: Illustration of 2'-FL Crystallization

Use Seeding with a Crystalline Mass [130] The centrifuge mother liquor combined with diluted crystal mass recovered from washing the equipment of Example 8, which had a 2'-FL concentration of 82.5% [1], a concentration DiFL of 15.9% [1], and a lactose concentration of <0.1% [1], was evaporated to a DS of 74.7% [4] (Rotavapor R-153 evaporator). The syrup (2.455 g) was seeded with 83 g of mother liquor from Example 8 (which contained small crystals) at a temperature of 67 ° C (1.67 supersaturation [8]). The seeded syrup was boiled at a temperature of 66 to 68Ό and a pressure of 1.9 x 10 ⁴ to 2.0 x 10 ⁴ Pa (190 to 200 mbar) for 4.1 h. The DS of the resulting crystal mass was 85.5% [3]. The centrifugation of a small sample of the dough without washing (Hettich Rotanta 460R centrifuge, 2,500 rpm, 10 min) provided a crystalline cake that has a purity of 2'-FL of 95.7% [1] and a purity of 2'- FL of mother liquor of 61.8% [1]. This corresponds to 71% yield of 2'-FL [2], [131] The resulting crystal mass was kept in a rotating Rotavapor bottle at a temperature of 66Ό overnight (17 h). After 21 h of sowing, centrifugation of a small sample of the dough without washing (Hettich Rotanta 460R centrifuge,

2,500 rpm, 10 min) provided a crystalline cake that has a purity of 2'-FL of 94.0% [1] and a purity of 2'-FL of mother liquor of 54.0% [1]. This corresponds to a 2'-FL yield of 81% [2]. The crystal size of the main crystal population was 80 to 200 pm [6].

[132] The resulting crystal mass was centrifuged with washing water in an amount equal to 71 ml / kg of mass DS (batch centrifuge, 22.5 cm basket diameter, 3,500 rpm, 5

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50/57 min). The moisture content of the combined non-dried cake was 3.4% [5]. The 2'-FL purity of the centrifuged and dried crystals (600, 2 h) was 100.0% [1]. The color was 35 ICUMSA [7] and the humidification content was 0.2% [3] ·

Example 10 Illustration of 2'-FL Crystallization from a High Lactose Solution and Using Seeding with a Crystalline Mass [133] An aqueous feed solution, which had a 2'-FL concentration of 84, 5% [1], a DiFL concentration of 7.9% [1], and a lactose concentration of 6.0% [1], was evaporated at a DS of 69.6% [3] (Rotavapor R evaporator -153). The solution was divided into two parts: one (1.394 g) was used to produce seeding mass, and the other was used as a real feed solution (10.640 g).

[134] The seeding dough syrup was seeded with 0.5 g of dry 2'-FL seed (crystals of Example 3) at a temperature of 68Ό (1.32 supersaturation [8]). The seeded syrup was boiled at a temperature of 65 to 68Ό and a pressure of 1.9 x 10 ⁴ Pa (190 mbar) for 5 h. The sowing mass obtained, which had a DS of 84.5% [3], was kept in a rotating Rotavapor bottle at a temperature of 55Ό overnight (7 pm).

[135] After 24 h of sowing the seeding mass, the actual feed solution was mixed with the 65Ό seeding mass (supersaturation of 1.32 [8]). The syrup was boiled at a temperature of 68Ό and pressure from 190 to 200 mbar for 9.6 h. The resulting sowing mass, which had a DS of 80.7% [3], was kept in a rotavapor bottle rotating at 68Ό overnight (16 h).

[136] After 24 h of sowing real feed solution, centrifuging a small sample of the unwashed mass (Hettich Rotanta 460R centrifuge, 2,500 rpm, 10 min) provided a cake

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[137] The crystal mass was centrifuged in 6 batches of washing water at an amount equal to 68 to 83 ml / kg of DS mass (batch centrifuge, basket diameter 22.5 cm, 3,500 rpm, 5 min). The average 2'-FL centrifugation yield for the 6 batches was 56% [2]. The average 2'-FL purity of the centrifuged and dried crystals (18 h at 40Ό and 1 h at 60Ό) was 99.0% [1], the melting point was 234.4 to 235.9 ° C [9], at average color was 63 ICUMSA [7], and the humidification content was 0.6% [3]. The purity of 2'-FL of mother liquor was 71.0% [1] and The color was 850 ICUMSA [7],

Example 11: Illustration of 2'-FL Crystallization

Of Mother Liquor and Equipment Wash of Example 10 [138] The mother liquor of centrifugation combined with diluted crystal mass recovered from washing the equipment of Example 10, which had a 2'-FL concentration of 72.4% [ 1], a DiFL concentration of 14.0% [1], and a lactose concentration of 10.6% [1], was evaporated to a DS of 70.3% [3] (Rotavapor R-153 evaporator) . The syrup (6.119 g) was seeded with 1.767 g of crystal mass from Example 10 at a temperature of 65Ό (1.19 supersaturation [8]). The seeded syrup was boiled at a temperature of 67 to 71Ό and a pressure of 1.8 x 10 ⁴ to 1.9 x 10 ⁴ Pa (180 to 190 mbar) for 5.4 h. The DS of the resulting crystal mass was 85.1% [3].

[139] The resulting crystal mass was kept in a rotating Rotavapor bottle at a temperature of 68Ό overnight (6 pm). After 24 h of sowing, centrifugation of a small sample of the dough without washing (Hettich Rotanta 460R centrifuge,

2,500 rpm, 10 min) provided a crystalline cake that has a purity of

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2'-FL of 94.1% [1] and a purity of 2'-FL of mother liquor of 46.5% [1]. This corresponds to 76% yield of 2'-FL [2]. The crystal size of the main crystal population was 100 to 200 pm [6].

[140] The crystal mass was centrifuged in 4 batches of washing water in an amount equal to 70 to 87 ml / kg of DS mass (batch centrifuge, basket diameter 22.5 cm, 3,500 rpm, 10 min ). The average 2'-FL centrifugation yield was 55% [2]. The average 2'-FL purity of the centrifuged and dried crystals (18 h at 40Ό and 1 h at 60Ό) was 97.9% [1]. The color was 204 ICUMSA [7] and the humidification content was 0.8% [3]. The purity of 2'-FL of mother liquor was 59.4% [1] and the color was 1409 ICUMSA [7],

Example 12: Illustration of 2'-FL Recrystallization [141] The 2'-FL crystals (1.842 g) from Example 11, which had a 97.9% 2'-FL concentration [1], a DiFL concentration 0.9% [1], and a lactose concentration of 1.3% [1], were dissolved in deionized water. The resulting solution was evaporated at a DS of 65.1% [3] (Rotavapor R-153 evaporator), and seeded with 0.88 g of dry 2'-FL seed (crystals of Example 3) at a temperature of 600 (1.30 supersaturation [8]). The seeded syrup was boiled at a temperature of 64 to 650 and a pressure of 1.8 x 10 ⁴ Pa (180 mbar) for 4.7 h. The DS of the resulting crystal mass was 77.8% [3].

[142] The resulting crystal mass was kept in a rotating Rotavapor bottle at a temperature of 640 overnight (7 pm). After 24 h of sowing, centrifugation of a small sample of the dough without washing (Hettich Rotanta 460R centrifuge,

2,500 rpm, 10 min) provided a crystalline cake that has a purity of 2'-FL of 99.7% [1] and a purity of 2'-FL of mother liquor of 95.5% [1]. This corresponds to 55% yield of 2'-FL [2]. The crystal size of the main crystal population was 30 to 100 pm [6].

[143] The crystal mass was centrifuged with washing water in

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53/57 an amount equal to 71 ml / kg of dough mass (batch centrifuge, basket diameter 22.5 cm, 3,500 rpm, 5 min). The moisture content of the combined non-dried cake was 5.8% [5]. The 2'-FL purity of the centrifuged and dried crystals (21 h at 40Ό and 1 h at 60Ό) was 100.0% [1]. The color was 142 ICUMSA [7] and the humidification content was 0.1% [3] ·

Example 13: Illustration of 2'-FL Crystallization [144] An aqueous feed syrup, which had a 2'-FL concentration of 89.6% [1], a DiFL concentration of 8.8% [1 ], and a lactose concentration of <0.1% [1], was evaporated at a DS of 67.5% [3] (Rotavapor R-153 evaporator). The resulting syrup (1.343 g) was seeded with 0.44 g of 2'-FL dry seed (crystals of Example 3) at a temperature of 61 ° (1.32 supersaturation [8]). The seeded syrup was boiled at a temperature of 64 to 65Ό and a pressure of 1.8 x 10 ⁴ Pa (180 mbar) for 5.5 h. The DS of the resulting crystal mass was 81.4% [3].

[145] The resulting crystal mass was kept in a rotating Rotavapor bottle at a temperature of 66Ό overnight (23 h). After 26 h of sowing, centrifugation of a small sample of the mass without washing (Hettich Rotanta 460R centrifuge,

2,500 rpm, 10 min) provided a crystalline cake that has a 2'-FL purity of 98.1% [1] and a 2'-FL purity of mother liquor of 76.1% [1]. This corresponds to 67% yield of 2'-FL [2]. The crystal size of the main crystal population was 30 to 100 pm [6].

[146] The crystal mass was centrifuged with washing water in an amount equal to 75 ml / kg of mass DS (batch centrifuge, basket diameter 22.5 cm, 3,500 rpm, 5 min). The moisture content of the combined non-dried cake was 4.6% [5]. The 2'-FL purity of the centrifuged and dried crystals (19 h at 40Ό and 1 h at 60Ό) was 100.0% [1]. The melting point was 233.2 to 234.1 ° C [9], the color was 47

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ICUMSA [7], and the humidification content was 0.1% [3].

Example 14: Illustration of 2'-FL Crystallization from a Lower 2'-FL Solution [147] An aqueous feed solution, which had a 2'-FL concentration of 60.5% [10], a concentration DiFL of 20.7% [10], and a lactose concentration of 15.1% [10], was evaporated at a DS of about 75%. The syrup was then seeded with dry seed crystals of 2'-FL in an amount equal to about 0.18 g / kg of syrup DS at a temperature of about 65Ό (supersaturation of about 1.3 [ 8]). The mixing was continued at this temperature for about 0.5 h. After that, the seeded syrup was evaporated at a DS of about 81.5% in about 20 h at a temperature of 65 to 70 °.

[148] The resulting crystal mass was kept in a mixture at 75 Ό for about 6 h, and then centrifuged with washing water in an amount equal to about 60 ml / kg of mass DS. The average 2'-FL centrifugation yield was 17.2%, and the average 2'-FL purity of the centrifuged crystals was 97.4% [10].

Example 15: Solubility of 2'-FL in Water [149] The dry 2'-FL crystals of Example 9 and deionized water were weighed in a 100 ml laboratory bottle. The container was tempered to a specific temperature (25, 40, 50, 60, or 700) using a water bath with thermostat. The contents were stirred continuously with a magnetic stirrer for at least 16 h to achieve the solubility balance. The 2'-FL crystals were added during the experiment, so that the crystal content in the equilibrium was around 10 to 20% by volume.

[150] After the system reached equilibrium, the mother liquor was separated from the crystals by sedimentation. The 2'-FL concentration of the mother liquor was determined by Karl Fischer titration. The results

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Table 2

Solubility of 2'-FL in water

temperature solubility Ό g of 2'-FL / 100 g of water 25 141 40 133

137

139

149

Example 16: Preparation of Amorphous 2'-FL from 2'-FL Crystalline [151] A solution of crystalline 2'-FL dissolved in water was spray-dried to obtain amorphous 2'-FL in powder form. The 2'-FL solution had the characteristics shown in Table 3.

Table 3

Parameter Value Total amount 826 kg Brix 47.48% Brix % calculated solids 45.5% pH 8.15 Conductivity 2 pS / cm Protein concentration 2.69 mg / L Temperature -30Ό

[152] A SPX Anhydro CSD Type 70 sprinkler dryer (vol. = ~ 43 m ³ ) (Soeborg, Denmark) equipped with an atomizer wheel was used to spray dry the 2'-FL solution. The 2'-FL solution was fed into the spray dryer at an initial rate of 79 kg / h, which was increased to 115 kg / h over the course of the first 3 hours of spray drying. The spray dryer definitions were as shown in Table 4.

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Table 4

Parameter Value Atomizer speed 25,136 rpm Air inlet flow 3,000 m ³ / h Air inlet temperature 135 G Air outlet temperature 104 <C

The drying process took 457 minutes.

[153] The dried 2'-FL powder obtained had the characteristics shown in Table 5.

Table 5

Parameter Value Total amount 340.1 kg Product yield 90.5% Loose product apparent density 601 g / l 100x compacted bulk density 772 g / l 625x compacted bulk density 832 g / l Humidification content (by weight) 2.12 to 2.21%

[154] The apparent density of the 70 g of powder was measured using a Jolting Stampf Volumeter (STAV 203, J. Engelsmann AG), a 250 ml measuring cylinder and a technical scale. The moisture content of the powder was measured using Karl-Fischer titration.

[155] The words understand, understand and understand must be interpreted inclusive rather than exclusively. This interpretation is intended to be the same as the interpretation that these words receive under United States patent law at the time of filing.

[156] The singular forms one and one are intended to include referents in the plural unless the context dictates otherwise. Thus, for example, a reference to the presence of a microorganism does not exclude the presence of multiple microorganisms to

Petition 870190087008, of 9/5/2019, p. 86/107

57/57 unless the context dictates otherwise.

[157] Any reference cited in that specification is incorporated by reference into this specification.

Claims (20)

1. Process to produce crystalline 2'-fucosyl-lactose from an initial aqueous solution comprising 2'-fucosyl-lactose and at least one other carbohydrate, characterized by the fact that: the process comprises: concentrating the initial solution to a supersaturated state in relation to 2'-fucosyl-lactose, and precipitating a crystal of 2'-fucosyl-lactose from the supersaturated solution while submitting the supersaturated solution to a temperature greater than 60Ό; and the supersaturated solution comprises at most 1% (by weight) of organic solvent during the precipitation of the 2'fucosyl-lactose crystal. 2. Process, according to claim 1, characterized by the fact that: the at least one other carbohydrate comprises difucosillactose, and the starting solution has a dry solids content with a concentration of difucosyl-lactose of at least 1% (where the percentage corresponds to a normalized peak area concentration obtained with the use of high performance liquid chromatography). 3. Process according to any of the preceding claims, characterized by the fact that: the at least one other carbohydrate comprises lactose, and the initial solution has a dry solids content with a lactose concentration of at least 0.1% (where the percentage corresponds to a normalized peak area concentration obtained with the use of high performance liquid chromatography). 4. Process, according to any of the Petition 870190087008, of 9/5/2019, p. 102/107 2/6 preceding claims, characterized by the fact that no organic solvent is added to the supersaturated solution while the supersaturated solution is subjected to a temperature greater than 60Ό. 5. Process according to any of the preceding claims, characterized by the fact that the initial solution is derived from a fermentation. Process according to any one of the preceding claims, characterized in that no organic solvent is added to the initial solution before or during the concentration of the initial solution to a supersaturated state. 7. Process for producing crystalline 2'-fucosyl-lactose from an initial aqueous solution comprising 2'-fucosyl-lactose and at least one other carbohydrate, characterized by the fact that: the process comprises: concentrating the initial solution to a supersaturated state in relation to 2'-fucosyl-lactose, and precipitating a 2'-fucosyl-lactose crystal from the supersaturated solution while submitting the supersaturated solution to a temperature of at least 40Ό; the initial solution has a dry solids content with a concentration of 2'-fucosyl-lactose from 70 to 95% (where the percentage corresponds to a normalized peak area concentration obtained using high performance liquid chromatography); and the supersaturated solution comprises at most 1% (by weight) of organic solvent during the precipitation of the 2'fucosyl-lactose crystal. 8. Process for producing crystalline 2'-fucosyl-lactose from an initial aqueous solution comprising 2'-fucosyl-lactose and at least one other carbohydrate, characterized by the fact that: Petition 870190087008, of 9/5/2019, p. 103/107 3/6 the process comprises: concentrating the initial solution to a supersaturated state in relation to 2'-fucosyl-lactose, and precipitating a 2'-fucosyl-lactose crystal from the supersaturated solution while submitting the supersaturated solution to a temperature of at least 40Ό; the initial solution has a dry solids content with a concentration of 2'-fucosyl-lactose of less than 98% (where the percentage corresponds to a normalized peak area concentration obtained using high performance liquid chromatography) ; and the supersaturated solution comprises at most 1% (by weight) of organic solvent during the precipitation of the 2'fucosyl-lactose crystal. 9. Process for producing crystalline 2'-fucosyl-lactose from an initial aqueous solution comprising 2'-fucosyl-lactose and at least one other carbohydrate, characterized by the fact that: the process comprises: concentrating the initial solution to a supersaturated state in relation to 2'-fucosyl-lactose, and precipitating a 2'-fucosyl-lactose crystal from the supersaturated solution while submitting the supersaturated solution to a temperature of at least 40Ό; the supersaturated solution comprises a maximum of 1% (by weight) of organic solvent during the precipitation of the 2'fucosyl-lactose crystal, and the crystalline 2'-fucosyl-lactose has a melting point of about 230 to about 239 Ό (as determined with a heating rate of 1 Ό / min using the European Pharmacopoeia capillary melting point method). Petition 870190087008, of 9/5/2019, p. 104/107 4/6 10. Process for producing crystalline 2'-fucosyl-lactose from an initial aqueous solution comprising 2'-fucosyl-lactose and at least one other carbohydrate, characterized by the fact that: the process comprises: concentrating the initial solution to a supersaturated state in relation to 2'-fucosyl-lactose, and precipitating a 2'-fucosyl-lactose crystal from the supersaturated solution while submitting the supersaturated solution to a temperature of at least 40 Ό; the supersaturated solution comprises at most 1% (by weight) of organic solvent during the precipitation of the 2'fucosyl-lactose crystal, and the crystalline 2'-fucosyl-lactose exhibits a powder X-ray diffraction reflection based on in a measurement using CuKa radiation, at 16.98 ± 0.20, 13.65 ± 0.20 and 18.32 ± 0.20 2Θ angles. 11. Process for producing amorphous 2'-fucosyl lactose, characterized by the fact that the process comprises: producing crystalline 2'-fucosyl-lactose according to the process according to any of claims 1 to 10, dissolving crystalline 2'-fucosyl-lactose in a solvent to form a purified solution of 2'-fucosyl-lactose, and precipitating Amorphous 2'-fucosyl-lactose from the purified 2'-fucosyl-lactose solution. Process according to claim 11, characterized in that the precipitation of amorphous 2'-fucosyl lactose from the purified 2'-fucosyl lactose solution comprises spray drying. 13. Process for producing a food, dietary supplement or medicine, characterized by the fact that the process comprises: Petition 870190087008, of 9/5/2019, p. 105/107 5/6 producing crystalline 2'-fucosyl-lactose according to the process, as defined in any one of claims 1 to 10, and both: mixing crystalline 2'-fucosyl-lactose with one or more ingredients suitable for the food, dietary supplement or medication; or dissolve the crystalline 2'-fucosyl-lactose in a solvent and mix the dissolved 2'-fucosyl-lactose with one or more ingredients suitable for the food, dietary supplement or medication. 14. Process for producing a food, dietary supplement or medication, characterized by the fact that the process comprises: produce amorphous 2'-fucosyl-lactose according to the process, as defined in claim 11 or 12, and both: mixing amorphous 2'-fucosyl lactose with one or more ingredients suitable for the food, dietary supplement or medication; or dissolve the amorphous 2'-fucosyl-lactose in a solvent and mix the dissolved 2'-fucosyl-lactose with one or more ingredients suitable for the food, dietary supplement or medication. 15. Process for producing infant formula, characterized by the fact that the process comprises: produce crystalline 2'-fucosyl-lactose according to the process, as defined in any one of claims 1 to 10, and both: mix the crystalline 2'-fucosyl-lactose with one or more ingredients of infant formula, or dissolve the crystalline 2'-fucosyl-lactose in a solvent and mix the dissolved 2'-fucosyl-lactose with one or more ingredients of Petition 870190087008, of 9/5/2019, p. 106/107 6/6 infant formula. 16. Process to produce an infant formula, characterized by the fact that the process comprises: produce amorphous 2'-fucosyl-lactose according to the process, as defined in claim 11 or 12, and both: mixing the amorphous 2'-fucosyl-lactose with one or more infant formula ingredients, or dissolving the amorphous 2'-fucosyl-lactose in a solvent and mixing the dissolved 2'-fucosyl lactose with one or more infant formula ingredients. 17. Crystalline 2'-fucosyl-lactose product, characterized by the fact that it is obtained from a process, as defined in any one of claims 1 to 10. 18. Amorphous 2'-fucosyl-lactose, characterized by the fact that it is obtained from a process, as defined in claim 11 or 12. 19. Food, dietary supplement or medication, characterized by the fact that it is obtained from a process, as defined in claim 13 or 14. 20. Infant formulation, characterized by the fact that it is obtained from a process, as defined in claim 15 or 16.